Phytotaxa 257 (1): 089–097 ISSN 1179-3155 (print edition) http://www.mapress.com/j/pt/ PHYTOTAXA Copyright © 2016 Magnolia Press Article ISSN 1179-3163 (online edition) http://dx.doi.org/10.11646/phytotaxa.257.1.7

Morphological and molecular identification of a new species of Truncospora (, ) in North America

CHANG-LIN ZHAO, Feng Xu & Donald H. Pfister* Farlow Herbarium of Cryptogamic Botany, Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, MA 02138, USA * Corresponding author’s e-mail: [email protected]

Abstract

Truncospora wisconsinensis sp. nov., a new poroid wood-inhabiting species, is proposed based on a combination of molecu- lar and morphological data. This species demonstrates a unique combination of characters including: annual habit; pileate basidiomata with a white pileus and pore surface; a dimitic hyphal system with non- to slightly dextrinoid, cyanophilous skeletal hyphae; and ellipsoid, truncate, slightly thick-walled, strongly dextrinoid basidiospores. Phylogenetic analyses us- ing ITS and partial tef1-α support the position of this new species as a sister clade of T. ohiensis.

Key words: , Phylogeny, Polypores, Taxonomy

Introduction

The Truncospora Pilát (1953) is characterized by relatively small, pileate basidiomata (about 1.5–3 cm long, 2.5–3.5 cm wide, and 1–4 cm thick), non-dextrinoid to dextrinoid skeletal hyphae, and truncate, strongly dextrinoid basidiospores (Pilát 1953, Corner 1989, Decock 2011, Zhao & Cui 2013, Spirin et al. 2015). The type species of Truncospora is T. ochroleuca (Berk.) Pilát. robledo et al. (2009), Zhao & Cui (2013), and Zhao et al. (2013) have carried out molecular studies, that indicate that Truncospora belongs to the core polyporoid clade, distinct from Perenniporia Murrill. Phylogenetic studies employing DNA sequence analyses of the ITS rDNA region (internal transcribed spacer ribosomal DNA) and TEF1 (translation elongation factor 1-α ) gene have been employed in phylogenic and diversity studies of the genus, which show that 7 species are recognized in the T. ohiensis group and 6 of them are described as new (Spirin et al. 2015). About 11 species are accepted in Truncospora worldwide (Pilát 1953, Corner 1989, Decock & Ryvarden 1999, Decock 2011, Zhao & Cui 2013, Spirin et al. 2015). Five are reported from the USA: T. arizonica Spirin & Vlasák, T. floridana Vlasák & Spirin, T. mexicana Vlasák, Spirin & Kou, T. ohiensis (Berk.) Pilát and T. tropicalis Vlasák & Spirin (Gilbertson & Ryvarden 1987, Spirin et al. 2015). A matching the concept of Truncospora was found by the senior author in the state of Wisconsin, USA. To confirm the affinity of this fungus, morphological as well as phylogenetic analyses were carried out using sequences of ITS rDNA and tef1-α.

Materials and methods

Morphological studies.—The specimens studied are deposited at the Farlow Herbarium, Harvard University (FH). Macro- morphological descriptions were based on field notes prepared at the time of collection. Color terms followed Petersen (1996). Microscopic measurements were made from slide preparations of dried specimens stained with Cotton Blue and Melzer’s reagent by light microscopy following Zhao et al. (2015). Sections were studied using an Olympus BX40 compound microscope. In presenting spore size variation, 5% of measurements were excluded from each end of the range and are given in parentheses. The following abbreviations are used: KOH = 5% potassium hydroxide, CB = Cotton Blue, CB+ = cyanophilous, IKI = Melzer’s reagent, IKI– = both non-amyloid and non-dextrinoid, L = indicates basidiospores

Accepted by Samantha Karunarathna: 18 Mar. 2016; published: 14 Apr. 2016 89 Licensed under a Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0 length (arithmetic average of all basidiospores), W = basidiospores width (arithmetic average of all basidiospores), Q = L/W ratio, n (a/b) = number of basidiospore (a) measured from given number of specimens (b).

TABLE 1. A list of species, specimens and GenBank accession number of sequences used in this study. Species name Sample no. Geographic origin GenBank accession no. References ITS TEF1 Perenniporia medulla-panis JV 0203/1 Czech Republic KJ410710 KJ410711 Spirin et al. 2015 Truncospora arizonica JV 1209/21 USA, Arizona KJ410695 KJ410715 Spirin et al. 2015 T. arizonica JV 1209/69 USA, Arizona KJ410696 KJ410716 Spirin et al. 2015 T. arizonica JV 1307/11J USA, Arizona KJ410697 KJ410717 Spirin et al. 2015 T. atlantica Spirin & Vlasák BRFM 1192 France GU731569 — Spirin et al. 2015 T. atlantica TAL128700 Spain, Canary Islands KJ410700 KJ410720 Spirin et al. 2015 T. atlantica JV 1311/4K Spain KJ410699 KJ410719 Spirin et al. 2015 T. detrita (Berk.) Decock MUCL 42649 French Guiana FJ411099 — Robledo et al. 2009 T. floridana JV 1008/78 USA, Florida KJ410704 KJ410723 Spirin et al. 2015 T. floridana JV 1008/85 USA, Florida KJ410705 KJ410724 Spirin et al. 2015 T. floridana JV 1008/86 USA, Florida KJ410706 KJ410725 Spirin et al. 2015 T. macrospora B.K. Cui China, Yunnan Zhao & Cui 2013 Yuan 3777 JX941574 — & C.L. Zhao T. macrospora Cui 8106 China, Yunnan JX941573 — Zhao & Cui 2013 T. mexicana JV 110983-1 USA, Texas KJ410708 KJ410727 Spirin et al. 2015 T. mexicana JV 110983-2 USA, Texas KJ410709 — Spirin et al. 2015 T. mexicana JV 0610/U Mexico, Veracruz KJ410707 KJ410726 Spirin et al. 2015 T. wisconsinensis C.L. Zhao USA, Wisconsin KP784666 In the present study CLZ 005 KP768408 & Pfister T. wisconsinensis CLZ 006 USA, Wisconsin KP768409 KP784667 In the present study T. wisconsinensis CLZ 007 USA, Wisconsin KP768410 KP784668 In the present study T. wisconsinensis CLZ 008 USA, Wisconsin KP768411 KP784669 In the present study T. ochroleuca JV 0610/7B Belize KJ410698 KJ410718 Spirin et al. 2015 T. ochroleuca MUCL 39563 Australia FJ411097 — Robledo et al. 2009 T. ohiensis JV1208/21 USA, Pennsylvania KJ410692 KJ410713 Spirin et al. 2015 T. ohiensis JV0309/114 USA, Pennsylvania KJ410694 — Spirin et al. 2015 T. ohiensis JV0509/64 USA, Tennessee KJ410693 KJ410714 Spirin et al. 2015 T. ohiensis MUCL 41036 USA FJ411096 — Robledo et al. 2009 T. ornata Spirin & Bukharova Cui 5714 China, Liaoning KF051056 — Zhao et al. 2013 T. ornata Dai 1644 China, Jilin KJ41069 — Spirin et al. 2015 T. ornata Spirin 6672 Russia, Khabarovsk KJ410690 KJ410712 Spirin et al. 2015 T. tropicalis JV 1008/45-1 USA, Florida KJ410702 — Spirin et al. 2015 T. tropicalis JV 1008/45-2 USA, Florida KJ410703 KJ410722 Spirin et al. 2015 T. tropicalis JV 1112/18J Puerto Rico, Rio Grande KJ410701 KJ410721 Spirin et al. 2015

90 • Phytotaxa 257 (1) © 2016 Magnolia Press ZHAO ET AL. DNA extraction, amplification, sequencing and phylogenetic analyses.—Genomic DNA was extracted from the specimens using the Qiagen DNeasy Plant Mini Kit (cat. no. 69104). 1/10 and 1/100 dilutions of the genomic DNA were used for PCR amplification of the ITS and tef1-α. The ITS region was amplified using primer pair ITS5 and ITS4 (White et al. 1990). The tef1-α gene was amplified with primer pair EF1-983F and EF1-2218R (Rehner & Buckley 2005). All PCR reactions were done in a Peltier Thermal cycler PTC-200 (MJ Research, Watertown, MA, USA) and used EconoTaq DNA Polymerase (Lucigen, Middleton, WI, USA). PCR amplification, purification, and sequencing were as previously described in Hansen et al. (2005) and Spirin et al. (2015). All newly generated sequences have been deposited in GenBank (Table 1). Sequencher 4.6 (GeneCodes, Ann Arbor, MI, USA) was used to edit the DNA sequence. Sequences generated in this study were aligned with sequences of the ten Truncospora species available from GenBank (Table 1) using ClustalX (Thompson et al. 1997) and manually adjusted in BioEdit (Hall 1999). In all phylogenetic analyses the sequence of Perenniporia medulla-panis (Jacq.) Donk (Table 1) was used as the outgroup to root tree following Spirin et al. (2015). The sequence alignment was deposited inTreeBase (submission ID 17104).

FIGURE 1. Maximum parsimony strict consensus tree illustrating the phylogeny of Truncospora wisconsinensis and related species based on ITS rDNA sequences. Branches are labeled with maximum likelihood bootstrap higher than 70%, parsimony bootstrap proportions higher than 50% and Bayesian posterior probabilities more than 0.95 respectively.

Approaches to phylogenetic analysis followed Zhao et al. (2015). Maximum parsimony (MP) analysis was applied to the dataset and the tree construction procedure was performed in PAUP* version 4.0b10 (Swofford 2002). All characters were equally weighted and gaps were treated as missing data. Trees were inferred using the heuristic search option with TBR branch swapping and 1000 random sequence additions. Max-trees were set to 5000, branches of zero a new species of Truncospora in America Phytotaxa 257 (1) © 2016 Magnolia Press • 91 length were collapsed and all parsimonious trees were saved. Clade robustness was assessed using a bootstrap (BT) analysis with 1000 replicates (Felsenstein 1985). Descriptive tree statistics, tree length (TL), consistency index (CI), retention index (RI), rescaled consistency index (RC), and homoplasy index (HI) were calculated for each Maximum Parsimonious Tree (MPT) generated. Sequences were also analyzed using Maximum Likelihood (ML) with RAxML- HPC2 on Abe through the Cipres Science Gateway (www.phylo.org; Miller et al. 2009). Branch support for ML analysis was determined by 1000 bootstrap replicate. MrModeltest 2.3 (Posada & Crandall 1998, Nylander 2004) was used to determine the best-fit evolution model for each data set for Bayesian inference (BI). BI was calculated with MrBayes3.1.2 with a general time reversible (GTR) model of DNA substitution and a gamma distribution rate variation across sites (Ronquist & Huelsenbeck 2003). Four Markov chains were run for 2 runs from random starting trees for 1 million generations (ITS) and for 500,000 generations (tef1-α). Trees were sampled every 100th generation. The first quarter of the generations were discarded as burn-in. A majority rule consensus tree of all remaining trees was calculated. Branches that received bootstrap support for maximum likelihood (BS) and Bayesian posterior probabilities (BPP) greater than or equal to 75 % (MP) and 0.95 (BPP) were considered as significantly supported, respectively.

Results

Molecular phylogeny The ITS dataset included sequences from 32 specimens representing 12 taxa. The dataset had an aligned length of 629 characters, of which 479 characters are constant, 63 parsimony-uninformative and 87 parsimony-informative. MP analysis yielded four equally parsimonious trees (TL = 239, CI = 0.749, HI = 0.251, RI = 0.896, RC = 0.671). Best model for ITS alignment estimated and applied in the BI was as follows: GTR+I+G, lset nst = 6, rates = invgamma; prset statefreqpr = dirichlet (1,1,1,1). BI resulted in a similar topology with an average standard deviation of split frequencies = 0.006038. Molecular phylogenetic analysis of the ITS sequences (Fig. 1) indicated that four specimens of the undescribed taxon grouped in a single clade, closely related to, but distinct from the T. ohiensis clade (100% BS, 96% MP, 1.00 BPP). Truncospora arizonica was the sister group to these two species (Fig. 1).

FIGURE 2. Maximum parsimony strict consensus tree illustrating the phylogeny of Truncospora wisconsinensis and related species based on tef1-α sequences. Branches are labeled with maximum likelihood bootstrap higher than 70%, parsimony bootstrap proportions higher than 50% and Bayesian posterior probabilities more than 0.95 respectively. Clade names follow Spirin et al. (2015).

the tef1-α dataset included sequences from 21 specimens representing 10 taxa. The dataset had an aligned length of 1115 characters, of which 862 characters are constant, 95 parsimony-uninformative and 158 parsimony-informative. MP analysis yielded eight equally parsimonious trees (TL = 396, CI = 0.778, HI = 0.222, RI = 0.870, RC = 0.678). Best model for tef1-α alignment was estimated and applied in the BI was as follows: GTR+I+G, lset nst = 6, rates =

92 • Phytotaxa 257 (1) © 2016 Magnolia Press ZHAO ET AL. invgamma; prset statefreqpr = dirichlet (1,1,1,1). BI resulted in a similar topology with an average standard deviation of split frequencies = 0.004582. the phylogeny of the tef1-α data set (Fig. 2) agreed with the ITS phylogeny. The four specimens of the new taxon formed a highly supported clade (100% BS, 96% MP and 1.00 BPP), with T. arizonica and T. ohiensis that were closely related.

Taxonomy

Truncospora wisconsinensis C.L. Zhao & Pfister, sp. nov. (Figs. 3, 4) MycoBankno.: MB 816122

Basidiocarps annual, pileate. Pileal surface white when fresh and white to pale whitish upon drying. Pore surface white; pores round. Hyphal system dimitic with subparallel to parallel, unbranched and slightly dextrinoid to non- dextrinoid skeletal hyphae. Basidiospores ellipsoid, hyaline, thick-walled, strongly dextrinoid, strongly cyanophilous, 9–11 × 6–7.5 µm.

FIGURE 3. Basidiomata of Truncospora wisconsinensis (drawn from the holotype). Scale bars: a= 2 cm; b = 1cm.

Type.—USA. Wisconsin, Dane County, Madison, Lakeshore Nature Reserve, alt. 263 m, on fallen trunk of Quercus alba, 10 December 2014, CLZhao 005/FH 00290969 (holotype, FH!). Etymology.—wisconsinensis referring to the locality of the type specimens.

a new species of Truncospora in America Phytotaxa 257 (1) © 2016 Magnolia Press • 93 FIGURE 4. Microscopic structures of Truncospora wisconsinensis (drawn from the holotype). a Basidiospores. b Basidioles. c Cystidioles. d Hyphae from trama. e Hyphae from context. Bars: a = 5 µm; b–e = 10 µm.

Basidiomata.—Annual, pileate, solitary or gregarious, without odor or taste and corky when fresh, becoming hard corky upon drying. Pilei dimidiate, projecting up to 1.5 cm, 2.5 cm wide, 1.2 cm thick at centre. Pileal surface pure white when fresh and white to pale whitish upon drying (59-60), smooth, gently concentrically sulcate, glabrous,

94 • Phytotaxa 257 (1) © 2016 Magnolia Press ZHAO ET AL. margin sharp. Pore surface pure white when fresh (60), white to grayish upon drying (59-60); pores round, 3–5 per mm; dissepiments thick, entire. Sterile margin narrow, white, up to 0.5 mm wide (60). Context white to pale cream (21, 60), up to 7 mm thick. Tubes concolorous with pore surface, up to 5 mm long. Hyphal structure.—Hyphal system dimitic; generative hyphae with clamp connections; skeletal hyphae non- to slightly dextrinoid, strongly cyanophilous; hyphae unchanged in KOH. Context trama.—Generative hyphae infrequent, hyaline, thin-walled, usually unbranched, 2–4 µm in diam; skeletal hyphae dominant, hyaline, thick-walled with a wide to narrow lumen, unbranched, subparallel to parallel, 3–5.5 µm in diam. Tube trama.—Generative hyphae infrequent, hyaline, thin-walled, usually unbranched, 2–3.5 µm in diam; skeletal hyphae dominant, hyaline, thick-walled with a wide to narrow lumen, unbranched, subparallel to parallel, 2.5–4 µm in diam. Hymenium.—Cystidia absent, but fusoid cystidioles present, hyaline, thin-walled, 15–20 × 3.5–5.5 µm; basidia not seen; basidioles predominant, barrel-shaped to pear-shaped; basidiospores ellipsoid, truncate, hyaline, slightly thick-walled, smooth, strongly dextrinoid, strongly cyanophilous, (8–)9–11(–11.5) × (5.5–)6–7.5(–8) µm, L = 9.8 µm, W = 6.58 µm, Q = 1.44–1.58 (n = 120/4). Additional specimens examined.—USA. Wisconsin, Dane County, Madison, Lakeshore Nature Reserve, alt. 263 m, on fallen trunk of Quercus alba, 10 December 2014, CLZhao 006/FH 00290968, CLZhao 007/FH 00290967, CLZhao 008/FH 00290966 (paratype, FH!).

Discussion

Truncospora wisconsinensis is characterized by an annual basidiomata with a whitish pileus and pore surface, a dimitic hyphal system with non- to slightly dextrinoid skeletal hyphae, and ellipsoid, truncate, slightly thick-walled, strongly dextrinoid basidiospore. 9–11 × 6–7.5 µm. Phylogenetic analyses demonstrated that T. wisconsinensis (Figs. 1, 2) is closely related to T. ohiensis and T. arizonica. Morphologically, T. ohiensis differs from T. wisconsinensis in its ochraceous to brownish-black pileus, smaller pores (5–6 per mm), trimitic hyphal system in tubes with branched, densely interwoven skeletal hyphae and large, and very thick-walled basidiospores (9.6–13.2 × 6.4–9.3 µm, Spirin et al. 2015). Truncospora arizonica is distinguished from T. wisconsinensis by its black pileus, thin-walled pore dissepiments, branched, strong dextrinoid skeletal hyphae and larger basidiospores (11.7–15.6 × 7.1–9.5 µm, Spirin et al. 2015). Morphologically, Truncospora oboensis Decock has some similarities with T. wisconsinensis that includes the annual basidiomata with white pileus and similar pores (3–4 per mm); it differs in its angular pores with thin-walled dissepiments and larger basidiospores (11–14 × 6.5–8.5 µm, Decock 2011). Truncospora detrita may be confused with T. wisconsinensis since it has similar basidiospores (10.3–12.5 × 6.2–7.8 µm), but T. detrita can be distinguished from T. wisconsinensis by larger basidiomata and a dark reddish brown crust on the pileus surface (Decock & Ryvarden 1999). the genus Truncospora has been often treated as a synonym of Perenniporia (Ryvarden 1972, 1991, Gilbertson & Ryvarden 1987, Ryvarden & Melo 2014). Decock & Ryvarden (1999) concluded that Perenniporia detrita (Berk.) Ryvarden, P. ochroleuca (Berk.) Pilát and P. ohiensis (Berk.) Ryvarden formed a morphologically homogeneous alliance that should be recognized at the genus level for which the name Truncospora was available. These taxa differ from Perenniporia by having relatively small, pileate basidiomata, and ellipsoid, large, apically truncate and dextrinoid basidiospores. Molecular phylogenetic analysis based on the IST+LSU rDNA gene regions supported Truncospora as monophyletic and places the genus as a clade distinct from the Perenniporia sensu stricto clade (Zhao & Cui 2013). In the present study, based on the framework provided by Spirin et al. (2015), three additional species, T. detrita, T. macrospora and T. wisconsinensis, are included in the ITS analysis. Truncospora detrita forms a monophyletic entity with a low support. Although T. macrospora groups with T. ochroleuca, the former differs from T. ochroleuca by having annual basidiocarps with a distinct dark brownish crust and larger basidiospores (16.5–19.5 × 8–9.5 µm). Morphological characters are very important when delimiting taxa in Perenniporia s.l. in previous studies (Ryvarden 1972, 1991, Gilbertson & Ryvarden 1987, Decock & Ryvarden 1999, Decock 2001, Robledo et al. 2009, Zhao & Cui 2013). Spirin et al. (2015) employed tefl-α to study the T. ohiensis group; that dataset of tef1-α produced 7 strongly supported terminal clades. In the present study, Truncospora ohiensis group is easily separated 9 clades based on tef1-α sequence, so the tef1-α may be a potential marker to apply on Perenniporia s.l. complex species in future. a new species of Truncospora in America Phytotaxa 257 (1) © 2016 Magnolia Press • 95 Key to accepted species of Truncospora in USA

1. Pores 3–5 per mm...... 2 Pores 5–7 per mm...... 4 2. Pileus light brown to black, basidiospores >12 μm long...... Truncospora arizonica Pileus white to ochraceous, basidiospores < 12μm long...... 3 3. Pores round, dissepiments thick, skeletal hyphae unbranched, parallel...... Truncospora wisconsinensis Pores angular, dissepiments thin, skeletal hyphae branched, interwoven...... Truncospora tropicalis 4. Species temperate in distribution; basidiomata brown to black...... Truncospora ohiensis Species subtropical or tropical in distribution; basidiomata white to pale cream to ochraceous...... 5 5. Context thicker than the tube layer, Florida...... Truncospora floridana Context distinctly thinner than the tube layer, coastal Gulf of Mexico...... Truncospora mexicana

Acknowledgements

Special thanks to Drs. Shuang-Hui He (BJFC, China), Bao-Kai Cui (BJFC, China) and Li-Wei Zhou (IFP, China) for assistance in collecting specimens. We express our gratitude to Genevieve Lewis-Gentry (Harvard University, USA) for managing specimens and Katherine F. LoBuglio (Harvard University, USA) is warmly thanked for her valuable advice on molecular work. The work was supported by a grant from the China Scholarship Council (CSC) to C.L. Zhao.

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